Met Kinase Inhibitor Screening Assay Kit

Intended Use

The Met Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of Met for the rapid and sensitive evaluation of inhibitors or activators. The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in recombinant "Tyrosine kinase-substrate-1", which is efficiently phosphorylated by Met in vitro.

Applications of this kit include:
1) Screening inhibitors or activators of recombinant catalytic domain of Met.
2) Detecting the effects of pharmacological agents on recombinant catalytic domain of Met.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

Upon receipt store all components at 4°C.
Don't expose reagents to excessive light.

Performance Characteristics

The Met Kinase Inhibitor Screening Assay Kit has been shown to detect the kinase activity of recombinant catalytic domain of Met. The assay shows good linearity of sample response.

General Description

The MET protooncogene was discovered because of the ability of oncogenic Met to mediate chemically induced transformation of a human osteogenic sarcoma cell line. This receptor tyrosine kinase is synthesized as a single-chain precursor, which undergoes intracellular proteolytic cleavage at a basic amino acid site, yielding a disulfide-linked heterodimer. Its C-terminal, intracellular region contains a multifunctional docking site that binds to various signaling molecules.
The ligand of the Met receptor is HGF/scatter factor, known to stimulate invasive growth of epithelial cells. It is a multifunctional factor affecting a number of cell targets including epithelium, endothelium, myoblasts, spinal motor neurons, and hematopoietic cells. Signaling pathways activated by the HGF-Met interaction mediate cell adhesion and motility. These cellular phenotypes, coupled to tightly regulated changes in cell growth, morphology, and survival, define a general pattern of invasive growth that occurs widely in normal development.
In addition, Met is involved in malignant cell transformation. Increased Met expression has been found in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. Point mutations in MET have been identified in hereditary and sporadic papillary renal carcinomas (3-5), hepatocellular and gastric carcinomas, and head and neck squamous carcinomas, Numerous experimental and clinical data indicate a particular role of HGF and Met in tumor invasive growth, a stage of tumor progression leading to metastases. Dysregulation of Met activity in cells is thought to be a key event underlying tumor metastasis, and indeed, Met overexpression and hyperactivation are reported to correlate with metastatic ability of the tumor cells.

Measurement of Met Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of Met kinase activity involves incubation of the Met kinase sample with substrate, either a natural or synthetic polypeptide (such as poly[Glu,Tyr]4:1), in the presence of Mg2+, Mn2+ and 32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates hazardous radioactive waste, and depends on a radioisotope of short half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The Met Kinase Inhibitor Screening Assay Kit uses a horseradish peroxidase coupled anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-wells ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect kinase activity of recombinant Met catalytic domain.